The effects of radiation, chemotherapeutic agents and other chemicals on mammalian spermatogenic cells which may result in mutations or sterility will be examined using rodents (primarily mice) as model systems. The major effort will be devoted to measuring biochemical damage in the DNA of specific germinal cells and determining the role of such damage in producing genetic mutations. In addition, we shall investigate the survival of spermatogenic stem cells following treatment and determine the relationship between stem cell inactivation and long term loss of fertility. The biochemical alterations in DNA produced by mutagen treatment will be assayed in cells at each stage of spermatogenesis since differences in the amounts of damage or capacity for repair most likely exist between tesiticular cells at different stages of maturation. Existing techniques for separating testicular cells and nuclei into homogeneous populations at specific stages of development will be applied to this problem. In addition, further improvements in these cell separation methods will be made. DNA damage and repair will be assayed by measuring strand breaks, cross-links, aklylations and unscheduled DNA synthesis. Alterations in DNA content of individual spermatogenic cells may be measured by flow microfluorimetry. Quantitative survival of testicular stem cells after high radiation doses can be measured by a histological method. We propose to develop biochemical assays to simplify the measurement of stem cell survival and to extend the study of lower doses and to chemical treatments. The utlimate objectives are, using rodents treated with radiation and mutagenic chemicals, to (1) understand the biochemical mechanisms underlying mutagenesis and (2) quantitate the loss of testicular stem cells and its relation to fertility, in order to extrapolate or extend these data to man.